Human natural killer cell activating factor ii

ABSTRACT

The invention relates to NKAF II polypeptides, polynucleotides encoding the polypeptides, methods for producing the polypeptides, in particular by expressing the polynucleotides, and agonists and antagonists of the polypeptides. The invention further relates to methods for utilizing such polynucleotides, polypeptides, agonists and antagonists for applications, which relate, in part, to research, diagnostic and clinical arts.

FIELD OF THE INVENTION

[0001] This invention relates, in part, to newly identifiedpolynucleotides and polypeptides;

[0002] variants and derivatives of the polynucleotides and polypeptides;processes for making the polynucleotides and the polypeptides, and theirvariants and derivatives; agonists and antagonists of the polypeptides;and uses of the polynucleotides, polypeptides, variants, derivatives,agonists and antagonists. In particular, in these and in other regards,the invention relates to polynucleotides and polypeptides of humanNatural Killer Cell Activating Factor II, sometimes hereinafter referredto as “NKAF II”.

[0003] This application claims benefit of 35 U.S.C. 120 based oncopending U.S. Provisional Application Ser. No. 60/014796, filed on Apr.3, 1996.

BACKGROUND OF THE INVENTION

[0004] The polypeptide of the present invention is a member of thenatural killer cell activating factor (NKAF) family and shows homologyto NKAF and human eosinophil major basic protein. Natural killer cells(NK cells) show a destructive effect on specific cancer cells.Lymphokines affecting the activity of these NK cells have thereforeattracted attention. It is reported that interleukin-2 and interferonenhance the activity of NK cells (Herberman, R. B., et al., Immunol.Rev., 44:13 (1979); Vose, B. M., et al., J. Immunol, 130:768 (1983) andDomzig, W. et al., J. Immunol., 130:1970(1983)).

[0005] NK cells are proposed to function as natural surveillance todeter cancer development in the body (Whiteside, T. and Herberman, R.B., Clin. Immunol. Immunopathol, 58:1-23 (1989) and Trinchieri, G., Adv.Immunol., 47:187-376 (1989)). NK cells are also important in controllingviral infection and the regulation of hematopoiesis (Trichieri, G., Adv.Immunol., 47:187-376 (1989); Kiessling, R., et al., Eur. J. Immunol.,7:655-663 (1977)).

[0006] The following facts indicate that NK cells play important rolesin the host defense against cancer. Namely, a nude mouse lacking T cellsby having a high NK activity does not always suffer from spontaneous orchemically induced carcinogenesis at a high frequency (Rygaard, J. etal., Immunol. Rev., 28:43 (1975); Stutman, D., et al., Science, 183:534(1974)); and the metastasis of transplanted cancer cells is promoted ina beige mouse having T cells but a genetically low NK activity(Shimamura, K. and Tamaoki, K., Jikken Igaku, 2:398 (1984); James E.Talmadge, et al., Nature, 284:622 (1980)) and a mouse having anartificially lowered NK activity (Shimamura, supra).

[0007] Human eosinophil major basic protein (MBP) has a nearly identicalsequence to that of known natural killer cell activating factor I. HumanMBP is one of the principal mediators of injury to parasites in tissuesin allergic inflammation. MBP is stored in eosinophil crystalloidgranules and released with other granule constituents during eosinophilactivation. MBP has no recognized enzymatic activity but it is toxic forsome helminths (Ackerman, S. J. et al., Am. J. Trop. Med. Hyg.,34:735-745 (1985)) and mammalian cells (Barker, R. L. et al., J. Clin.Invest. 88:798-805(1991)) in vitro. MBP is expressedprincipallyinbone-marroweosinophils,but it is also synthesized in basophils andplacental trophoblast x-cells (Wasmoen, T. L. et al., J. Exp. Med.,170:2051-2063(1989)).

[0008] MBP stimulates the effector function of a wide variety of cells,namely MBP stimulates the noncytolytic release of histamine from humanbasophils and rat mass cells (O'Donnell, M. A. et al., J. Exp. Med.157:1981 (1983)). MBP also stimulates neutrophils to release superoxideand ion and lysozyme, but not beta-glucuronidase or lacticdehydrogenase, and MBP enhances the expression of CR3 and P150, 95 byneutrophils. This indicates that MBP activates other cells associatedwith inflammation, such as basophils, platelets and neutrophils. Theeffector mechanisms may play a role in pathophysiology of variousdiseases where granule proteins are released. For example, MBP has beenthought a cause for increased airway responsiveness, for example,bronchial asthma.

[0009] The effects of the natural killer cell activating factors arevaried and influence numerous functions, both normal and abnormal, inthe biological processes of the mammalian system. There is a clear need,therefore, for identification and characterization of proteins thatinfluence biological activity, both normally and in diseased states. Inparticular, there is a need to isolate and characterize additionalnatural killer cell activating factors akin to known natural killer cellactivating factors which may be employed, therefore, for preventing,ameliorating or correcting disfimctions or disease or augmentingpositive natural actions of such receptors.

SUMMARY OF THE INVENTION

[0010] Toward these ends, and others, it is an object of the presentinvention to provide polypeptides, inter alia, that have been identifiedas novel NKAF II by homology between the amino acid sequence set out inFIG. 1 (SEQ ID NO:1 and 2) and known amino acid sequences of otherproteins such as human eosinophil granule major basic protein.

[0011] It is a further object of the invention, moreover, to providepolynucleotides that encode NKAF II, particularly polynucleotidesthatencode the polypeptide herein designated NKAF II.

[0012] In a particularly preferred embodiment of this aspect of theinvention the polynucleotide comprises the region encoding human NKAF IIin the sequence set out in FIG. 1 (SEQ ID NO:2).

[0013] In accordance with this aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressed by the human cDNA contained in ATCC Deposit No. 97465.

[0014] In accordance with this aspect of the invention there areprovided isolated nucleic acid molecules encoding human NKAF II,including mRNAs, cDNAs, genomic DNAs and, in further embodiments of thisaspect of the invention, biologically, diagnostically, clinically ortherapeutically useful variants, analogs or derivatives thereof, orfragments thereof, including fragments of the variants, analogs andderivatives.

[0015] Among the particularly preferred embodiments of this aspect ofthe invention are naturally occurring allelic variants of human NKAF II.

[0016] It also is an object of the invention to provide NKAF IIpolypeptides, particularly human NKAF II polypeptides, that inhibit thegrowth of leukemia cells, to treat viral infection, to augment theeffects of natural killer protein to treat neoplasias such as tumors andcancers, to prevent inflammation, to treat parasitic infection, toregulate hematopoiesis, to prevent damage from superoxide radicals inthe body, for example, tissue injury and aging and to enhance animmunological response.

[0017] In accordance with this aspect of the invention there areprovided novel polypeptides of human origin referred to herein as NKAFII as well as biologically, diagnostically or therapeutically usefulfragments, variants and derivatives thereof, variants and derivatives ofthe fragments, and analogs of the foregoing.

[0018] Among the particularly preferred embodiments of this aspect ofthe invention are variants of human NKAF II encoded by naturallyoccurring alleles of the human NKAF II gene.

[0019] It is another object of the invention to provide a process forproducing the aforementioned polypeptides, polypeptide fragments,variants and derivatives, fragments of the variants and derivatives, andanalogs of the foregoing. In a preferred embodiment of this aspect ofthe invention there are provided methods for producing theaforementioned NKAF II polypeptides comprising culturing host cellshaving expressibly incorporated therein an exogenously-derivedhuman NKAFII-encoding polynucleotide under conditions for expression of human NKAFII in the host and then recovering the expressed polypeptide.

[0020] In accordance with another object the invention there areprovided products, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides for research,biological, clinical and therapeutic purposes, inter alia.

[0021] In accordance with certain preferred embodiments of this aspectof the invention, there are provided products, compositions and methods,inter alia, for, among other things: assessing NKAF II expression incells by determining NKAF II polypeptides or NKAF II-encoding mRNA;assaying genetic variation and aberrations, such as defects, in NKAF IIgenes; and administering a NKAF II polypeptide or polynucleotide to anorganism to augment NKAF II function or remediate NKAF II dysfunction.

[0022] In accordance with certain preferred embodiments of this andother aspects of the invention there are provided probes that hybridizeto human NKAF II sequences.

[0023] In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against NKAF II polypeptides. Incertain particularly preferred embodiments in this regard, theantibodies are highly selective for human NKAF II. In accordance withanother aspect of the present invention, there are provided NKAF IIagonists.

[0024] Among preferred agonists are molecules that mimic NKAF II, thatbind to NKAF II-binding molecules or receptor molecules, and that elicitor augment NKAF II-induced responses. Also among preferred agonists aremolecules that interact with NKAF II or NKAF II polypeptides, or withother modulators of NKAF II activities, and thereby potentiate oraugment an effect of NKAF II or more than one effect of NKAF II.

[0025] In accordance with yet another aspect of the present invention,there are provided NKAF II antagonists. Among preferred antagonists arethose which mimic NKAF II so as to bind to NKAF II receptor or bindingmolecules but not elicit a NKAF II-induced response or more than oneNKAF II-induced response. Also among preferred antagonists are moleculesthat bind to or interact with NKAF II so as to inhibit an effect of NKAFII or more than one effect of NKAF II or which prevent expression ofNKAF II.

[0026] The agonists and antagonists may be used to mimic, augment orinhibit the action of NKAF II polypeptides. They may be used, forinstance, to inhibit the action of such polypeptides, for example, toprevent allergic inflammation, hypersensitivity, bronchial asthma,eosinophilia, chronic urticaria, atopic dermatitis, Kimura's disease andbone marrow transplant rejection.

[0027] In a further aspect of the invention there are providedcompositions comprising a NKAF II polynucleotide or a NKAF IIpolypeptide for administration to cells in vitro, to cells ex vivo andto cells in vivo, or to a multicellular organism. In certainparticularly preferred embodiments of this aspect of the invention, thecompositions comprise a NKAF II polynucleotide for expression of a NKAFII polypeptide in a host organism for treatment of disease. Particularlypreferred in this regard is expression in a human patient for treatmentof a dysfunction associated with aberrant endogenous activity of NKAFII.

[0028] Other objects, features, advantages and aspects of the presentinvention will become apparent to those of skill from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following drawings depict certain embodiments of theinvention. They are illustrative only and do not limit the inventionotherwise disclosed herein.

[0030]FIG. 1 shows the nucleotide and deduced amino acid sequence ofhuman NKAF II.

[0031] The determined leader sequence is underlined.

[0032]FIG. 2 shows the regions of similarity between amino acidsequences of NKAF II and human eosinophil granule major basic proteinpolypeptides.

[0033]FIG. 3 shows structural and functional features of NKAF II deducedby the indicated techniques, as a function of amino acid sequence.

GLOSSARY

[0034] The following illustrative explanations are provided tofacilitate understanding of certain terms used frequently herein,particularly in the examples. The explanations are provided as aconvenience and are not limitative of the invention.

[0035] DIGESTION of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes referred to herein are commerciallyavailable and their reaction conditions, cofactors and otherrequirements for use are known and routine to the skilled artisan.

[0036] For analytical purposes, typically, 1 mg of plasmid or DNAfragment is digested with about 2 units of enzyme in about 20 ml ofreaction buffer. For the purpose of isolating DNA fragments for plasmidconstruction, typically 5 to 50 mg of DNA are digested with 20 to 250units of enzyme in proportionately larger volumes.

[0037] Appropriate buffers and substrate amounts for particularrestriction enzymes are described in standard laboratory manuals, suchas those referenced below, and they are specified by commercialsuppliers.

[0038] Incubation times of about 1 hour at 37° C. are ordinarily used,but conditions may vary in accordance with standard procedures, thesupplier's instructions and the particulars of the reaction. Afterdigestion, reactions may be analyzed, and fragments may be purified byelectrophoresis through an agarose or polyacrylamide gel, using wellknown methods that are routine for those skilled in the art.

[0039] GENETIC ELEMENT generally means a polynucleotide comprising aregion that encodes a polypeptide or a region that regulatestranscription or translation or other processes important to expressionof the polypeptide in a host cell, or a polynucleotide comprising both aregion that encodes a polypeptide and a region operably linked theretothat regulates expression.

[0040] Genetic elements may be comprised within a vector that replicatesas an episomal element; that is, as a molecule physically independent ofthe host cell genome. They may be comprised within mini-chromosomes,such as those that arise during amplification of transfected DNA bymethotrexate selection in eukaryotic cells. Genetic elements also may becomprised within a host cell genome; not in their natural state but,rather, following manipulation such as isolation, cloning andintroduction into a host cell in the form of purified DNA or in avector, among others.

[0041] ISOLATED means altered “by the hand of man” from its naturalstate; i.e., that, if it occurs in nature, it has been changed orremoved from its original environment, or both.

[0042] For example, a naturally occurring polynucleotide or apolypeptide naturally present in a living animal in its natural state isnot “isolated,” but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is “isolated”, as theterm is employed herein. For example, with respect to polynucleotides,the term isolated means that it is separated from the chromosome andcell in which it naturally occurs.

[0043] As part of or following isolation, such polynucleotides can bejoined to other polynucleotides, such as DNAs, for mutagenesis, to formfusion proteins, and for propagation or expression in a host, forinstance. The isolated polynucleotides, alone or joined to otherpolynucleotides such as vectors, can be introduced into host cells, inculture or in whole organisms. Introduced into host cells in culture orin whole organisms, such DNAs still would be isolated, as the term isused herein, because they would not be in their naturally occurring formor environment. Similarly, the polynucleotides and polypeptides mayoccur in a composition, such as a media formulations, solutions forintroduction of polynucleotides or polypeptides, for example, intocells, compositions or solutions for chemical or enzymatic reactions,for instance, which are not naturally occurring compositions, and,therein remain isolated polynucleotides or polypeptides within themeaning of that term as it is employed herein.

[0044] LIGATION refers to the process of forming phosphodiester bondsbetween two or more polynucleotides, which most often are doublestranded DNAs. Techniques for ligation are well known to the art andprotocols for ligation are described in standard laboratory manuals andreferences, such as, for instance, Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, New York (1989) and Maniatis et al., pg. 146, as citedbelow.

[0045] OLIGONUCLEOTIDE(S) refers to relatively short polynucleotides.Often the term refers to single-stranded deoxyribonucleotides, but itcan refer as well to single-or double-stranded ribonucleotides, RNA:DNAhybrids and double-stranded DNAs, among others.

[0046] Oligonucleotides, such as single-stranded DNA probeoligonucleotides, often are synthesized by chemical methods, such asthose implemented on automated oligonucleotide synthesizers. However,oligonucleotides can be made by a variety of other methods, including invitro recombinant DNA-mediated techniques and by expression of DNAs incells and organisms.

[0047] Initially, chemically synthesized DNAs typically are obtainedwithout a 5′ phosphate. The 5′ ends of such oligonucleotides are notsubstrates for phosphodiester bond formation by ligation reactions thatemploy DNA ligases typically used to form recombinant DNA molecules.Where ligation of such oligonucleotides is desired, a phosphate can beadded by standard techniques, such as those that employ a kinase andATP.

[0048] The 3′ end of a chemically synthesized oligonucleotide generallyhas a free hydroxyl group and, in the presence of a ligase, such as T4DNA ligase, readily will form a phosphodiester bond with a 5′ phosphateof another polynucleotide, such as another oligonucleotide. As is wellknown, this reaction can be prevented selectively, where desired, byremoving the 5′ phosphates of the other polynucleotide(s)prior toligation.

[0049] PLASMIDS generally are designated herein by a lower case ppreceded and/or followed by capital letters and/or numbers, inaccordance with standard naming conventions that are familiar to thoseof skill in the art. Starting plasmids disclosed herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids by routine application ofwell known, published procedures. Many plasmids and other cloning andexpression vectors that can be used in accordance with the presentinvention are well known and readily available to those of skill in theart. Moreover, those of skill readily may construct any number of otherplasmids suitable for use in the invention. The properties, constructionand use of such plasmids, as well as other vectors, in the presentinvention will be readily apparent to those of skill from the presentdisclosure.

[0050] POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide,which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single-and double-stranded DNA, DNA that is a mixtureof single-and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, polynucleotide as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.

[0051] As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotidesasthe term is used herein.

[0052] It will be appreciated that a great variety of modifications havebeen made to DNA and RNA that serve many useful purposes known to thoseof skill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

[0053] POLYPEPTIDES, as used herein, includes all polypeptides asdescribed below. The basic structure of polypeptides is well known andhas been described in innumerable textbooks and other publications inthe art. In this context, the term is used herein to refer to anypeptide or protein comprising two or more amino acids joined to eachother in a linear chain by peptide bonds. As used herein, the termrefers to both short chains, which also commonly are referred to in theart as peptides, oligopeptides and oligomers, for example, and to longerchains, which generally are referred to in the art as proteins, of whichthere are many types. It will be appreciated that polypeptides oftencontain amino acids other than the 20 amino acids commonly referred toas the 20 naturally occurring amino acids, and that many amino acids,including the terminal amino acids, may be modified in a givenpolypeptide, either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques which are well known to the art. Even the commonmodifications that occur naturally in polypeptides are too numerous tolist exhaustively here, but they are well described in basic texts andin more detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art.

[0054] Among the known modifications which may be present inpolypeptides of the present are, to name an illustrative few,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0055] Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Analysis for protein modifications andnonprotein cofactors, Meth. Enzymol. 182: 626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992).

[0056] It will be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptidesmay be branched as a result of ubiquitination, and they may be circular,with or without branching, generally as a result of posttranslationevents, including natural processing event and events brought about byhuman manipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationnatural process and by entirely synthetic methods, as well.

[0057] Modifications can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. In fact, blockage of the amino or carboxyl group in apolypeptide, or both, by a covalent modification, is common in naturallyoccurring and synthetic polypeptides and such modifications may bepresent in polypeptides of the present invention, as well. For instance,the amino terminal residue of polypeptides made in E. coli, prior toproteolytic processing, almost invariably will be N-formylmethionine.

[0058] The modifications that occur in a polypeptide often will be afunction of how it is made. For polypeptides made by expressing a clonedgene in a host, for instance, the nature and extent of the modificationsin large part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcell often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to express efficiently mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

[0059] It will be appreciated that the same type of modification may bepresent in the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications.

[0060] In general, as used herein, the term polypeptide encompasses allsuch modifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

[0061] VARIANT(S) of polynucleotides or polypeptides, as the term isused herein, are polynucleotides or polypeptides that differ from areference polynucleotide or polypeptide, respectively. Variants in thissense are described below and elsewhere in the present disclosure ingreater detail.

[0062] (1) A polynucleotide that differs in nucleotide sequence fromanother, reference polynucleotide. Generally, differences are limited sothat the nucleotide sequences of the reference and the variant areclosely similar overall and, in many regions, identical.

[0063] As noted below, changes in the nucleotide sequence of the variantmay be silent. That is, they may not alter the amino acids encoded bythe polynucleotide. Where alterations are limited to silent changes ofthis type a variant will encode a polypeptide with the same amino acidsequence as the reference. Also as noted below, changes in thenucleotide sequence of the variant may alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence, as discussed below.

[0064] (2) A polypeptide that differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference and the variant are closely similaroverall and, in many region, identical.

[0065] A variant and reference polypeptide may differ in amino acidsequence by one or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

[0066] RECEPTOR MOLECULE, as used herein, refers to molecules which bindor interact specifically with NKAF II polypeptides of the presentinvention, including not only classic receptors, which are preferred,but also other molecules that specifically bind to or interact withpolypeptides of the invention (which also may be referred to as “bindingmolecules” and “interaction molecules,” respectively and as “NKAF IIbinding molecules” and “NKAF II interaction molecules.” Binding betweenpolypeptides of the invention and such molecules, including receptor orbinding or interaction molecules may be exclusive to polypeptides of theinvention, which is very highly preferred, or it may be highly specificfor polypeptides of the invention, which is highly preferred, or it maybe highly specific to a group of proteins that includes polypeptides ofthe invention, which is preferred, or it may be specific to severalgroups of proteins at least one of which includes polypeptides of theinvention.

[0067] Receptors also may be non-naturally occurring, such as antibodiesand antibody-derived reagents that bind specifically to polypeptides ofthe invention.

DESCRIPTION OF THE INVENTION

[0068] The present invention relates to novel NKAF II polypeptides andpolynucleotides, among other things, as described in greater detailbelow. In particular, the invention relates to polypeptides andpolynucleotides of a novel human NKAF II, which is related by amino acidsequence homology to human eosinophil granule major basic protein. Theinvention relates especially to NKAF II having the nucleotide and aminoacid sequences set out in FIG. 1 (SEQ ID NO:1 and 2), and to the NKAF IInucleotide and amino acid sequences of the human cDNA in ATCC DepositNo. 97465 which is herein referred to as “the deposited clone” or as the“cDNA of the deposited clone.” It will be appreciated that thenucleotide and amino acid sequences set out in FIG. 1 (SEQ ID NO:2) wereobtained by sequencing the human cDNA of the deposited clone. Hence, thesequence of the deposited clone is controlling as to any discrepanciesbetween the two and any reference to the sequence of FIG. 1 (SEQ IDNO:1) includes reference to the sequence of the human cDNA of thedeposited clone.

[0069] Polynucleotides

[0070] In accordance with one aspect of the present invention, there areprovided isolated polynucleotides that encode the NKAF II polypeptidehaving the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2).

[0071] Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1 (SEQ ID NO:1), a polynucleotide of thepresent invention encoding a human NKAF II polypeptide may be obtainedusing standard cloning and screening procedures, such as those forcloning cDNAs using mRNA from cells of human tissue as startingmaterial. Illustrative of the invention, the polynucleotide set out inFIG. 1 (SEQ ID NO:1) was discovered in a cDNA library derived from cellsof human bone marrow and fetal liver.

[0072] Human NKAF II of the invention is structurally related to otherproteins of the human natural killer cell family, as shown by theresults of sequencing the human cDNA encoding human NKAF II in thedeposited clone. The human cDNA sequence thus obtained is set out inFIG. 1 (SEQ ID NO:1). It contains an open reading frame encoding aprotein of about 225 amino acid residues. NKAF II has a deducedmolecular weight of about 25.5 kDa; an isoelectric point of 4.661 and a−11.752 charge at pH of 7.0. The protein exhibits greatest homology tohuman eosinophil granule major basic protein among known proteins. NKAFII of FIG. 1 (SEQ ID NO:2) has about 65.8% similarity and about 50.2%identity with the amino acid sequence of human eosinophil granule majorbasic protein and NKAF as disclosed in U.S. Pat. No. 5,316,933.

[0073] The amino acid sequence of the complete NKAF II protein includesa leader sequence and a mature protein. More in particular, the presentinvention provides nucleic acid molecules encoding a mature form of theNKAF II protein. Thus, according to the signal hypothesis, once exportof the growing protein chain across the rough endoplasmic reticulum hasbeen initiated, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the complete polypeptideto produce a secreted “mature” form of the protein. Most mammalian cellsand even insect cells cleave secreted proteins with the samespecificity. However, in some cases, cleavage of a secreted protein isnot entirely uniform, which results in two or more mature species of theprotein. Further, it has long been known that the cleavage specificityof a secreted protein is ultimately determined by the primary structureof the complete protein, that is, it is inherent in the amino acidsequence of the polypeptide. Therefore, the present invention provides anucleotide sequence encoding the mature NKAF II polypeptide having theamino acid sequence encoded by the cDNA clone contained in the hostidentified as ATCC Deposit No. 97465. By the “mature NKAF II polypeptidehaving the amino acid sequence encoded by the cDNA clone in ATCC DepositNo. 97465” is meant the mature form(s) of the NKAF II protein producedby expression in a mammalian cell (e.g., COS cells, as described below)of the complete open reading frame encoded by the human DNA sequence ofthe clone contained in the vector in the deposited host.

[0074] In addition, methods for predicting whether a protein has asecretory leader as well as the cleavage point for that leader sequenceare available. For instance, the method of McGeoch (Virus Res. 3:271-286(1985)) uses the information from a short N-terminal charged region anda subsequent uncharged region of the complete (uncleaved) protein. Themethod of von Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) uses theinformation from the residues surrounding the cleavage site, typicallyresidues −13 to +2 where +1 indicates the amino terminus of the matureprotein. The accuracy of predicting the cleavage points of knownmammalian secretory proteins for each of these methods is in the rangeof 75-80% (von Heinje, supra). However, the two methods do not alwaysproduce the same predicted cleavage point(s) for a given protein.

[0075] In the present case, the deposited cDNA has been expressed in abaculovirus vector in insect cells as described hereinbelow, and aminoacid sequencing of the amino terminus of the secreted species indicatedthat the mature NKAF II protein comprises amino acids 1 to 208 of SEQ IDNO:2. Thus, the leader sequence of the NKAF II protein in the amino acidsequence of SEQ ID NO:2 is 17 amino acids, from position −17 to −1.

[0076] More in particular the invention includes polypeptides comprisingamino acids: 14 to 225, 15 to 225, 16 to 225, 17 to 225, 18 to 225, 19to 225, 20 to 225, 21 to 225, 22 to 225, and 23 to 225, all of SEQ IDNO:2. Polynucleotides encoding such polypeptides are also provided.

[0077] Polynucleotides of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance, cDNAand genomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

[0078] The coding sequence which encodes the polypeptide may beidentical to the coding sequence of the polynucleotide shown in FIG. 1(SEQ ID NO:1). It also may be a polynucleotide with a differentsequence, which, as a result of the redundancy (degeneracy) of thegenetic code, encodes the polypeptide of the DNA of FIG. 1 (SEQ IDNO:1).

[0079] Polynucleotides of the present invention which encode thepolypeptide of FIG. 1 (SEQ ID NO:2) may include, but are not limited tothe coding sequence for the mature polypeptide, by itself; the codingsequence for the mature polypeptide and additional coding sequences,such as those encoding a leader or secretory sequence, such as a pre-,or pro- or prepro- protein sequence; the coding sequence of the maturepolypeptide, with or without the aforementioned additional codingsequences, together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′0 and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing—including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; additionalcoding sequence which codes for additional amino acids, such as thosewhich provide additional functionalities. Thus, for instance, thepolypeptide may be fused to a marker sequence, such as a peptide, whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein.

[0080] The HA tag corresponds to an epitope derived of influenzahemagglutinin protein, which has been described by Wilson et al., Cell37: 767 (1984), for instance.

[0081] In accordance with the foregoing, the term “polynucleotideencoding a polypeptide” as used herein encompasses polynucleotides whichinclude a sequence encoding a polypeptide of the present invention,particularly the human NKAF II having the amino acid sequence set out inFIG. 1 (SEQ ID NO:2). The term encompasses polynucleotides that includea single continuous region or discontinuous regions encoding thepolypeptide (for example, interrupted by introns) together withadditional regions. The present invention further relates to variants ofthe herein above described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the deduced amino acidsequence of FIG. 1 (SEQ ID NO:2). A variant of the polynucleotide may bea naturally occurring variant such as a naturally occurring allelicvariant, or it may be a variant that is not known to occur naturally.Such non-naturally occurring variants of the polynucleotide may be madeby mutagenesis techniques, including those applied to polynucleotides,cells or organisms.

[0082] Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions, deletions or additions may involve one ormore nucleotides. The variants may be altered in coding or non-codingregions or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions.

[0083] Among the particularly preferred embodiments of the invention inthis regard are polynucleotides encoding polypeptides having the aminoacid sequence of NKAF II set out in FIG. 1 (SEQ ID NO:2); variants,analogs, derivatives and fragments thereof, and fragments of thevariants, analogs and derivatives.

[0084] Further particularly preferred in this regard are polynucleotidesencoding NKAF II variants, analogs, derivatives and fragments, andvariants, analogs and derivatives of the fragments, which have the aminoacid sequence of the NKAF II poLypeptide of FIG. 1 (SEQ ID NO: 2) inwhich several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acidresidues are substituted, deleted or added, in any combination.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of the NKAFII. Also especially preferred in this regard are conservativesubstitutions. Most highly preferred are polynucleotides encodingpolypeptides having the amino acid sequence of FIG. 1 (SEQ ID NO:2)without substitutions. Further preferred embodiments of the inventionare polynucleotides that are at least 70% identical to a polynucleotideencoding the NKAF II polypeptide having the amino acid sequence set outin FIG. 1 (SEQ ID NO:2), and polynucleotides which are complementary tosuch polynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identical toa polynucleotide encoding the NKAF II polypeptide and polynucleotidescomplementary thereto. In this regard, polynucleotides at least 90%identical to the same are particularly preferred, and among theseparticularly preferred polynucleotides, those with at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the more preferred.

[0085] Particularly preferred embodiments in this respect, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame biological function or activity as the mature polypeptide encodedby the human cDNA of FIG. 1 (SEQ ID NO:1).

[0086] The present invention further relates to polynucleotides thathybridize to the herein above-described sequences. In this regard, thepresent invention especially relates to polynucleotides which hybridizeunder stringent conditions to the herein above-describedpolynucleotides. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences.

[0087] As discussed additionally herein regarding polynucleotide assaysof the invention, for instance, polynucleotides of the invention asdiscussed above, may be used as a hybridization probe for cDNA andgenomic DNA to isolate full-length cDNAs and genomic clones encodingNKAF II and to isolate cDNA and genomic clones of other genes that havea high sequence similarity to the human NKAF II gene. Such probesgenerally will comprise at least 15 bases. Preferably, such probes willhave at least 30 bases and may have at least 50 bases. Particularlypreferred probes will have at least 30 bases and will have 50 bases orless.

[0088] For example, the coding region of the NKAF II gene may beisolated by screening using the known DNA sequence to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the present invention is then used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

[0089] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery oftreatments and diagnostics to human disease, as further discussed hereinrelating to polynucleotide assays, inter alia.

[0090] The polynucleotides may encode a polypeptide which is the matureprotein plus additional amino or carboxyl-terminal amino acids, or aminoacids interior to the mature polypeptide (when the mature form has morethan one polypeptide chain, for instance). Such sequences may play arole in processing of a protein from precursor to a mature form, mayfacilitate protein trafficking, may prolong or shorten protein half-lifeor may facilitate manipulation of a protein for assay or production,among other things. As generally is the case in situ, the additionalamino acids may be processed away from the mature protein by cellularenzymes.

[0091] A precursor protein, having the mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0092] In sum, a polynucleotide of the present invention may encode amature protein, a mature protein plus a leader sequence (which may bereferred to as a preprotein), a precursor of a mature protein having oneor more prosequences which are not the leader sequences of a preprotein,or a preproprotein, which is a precursor to a proprotein, having aleader sequence and one or more prosequences, which generally areremoved during processing steps that produce active and mature forms ofthe polypeptide.

[0093] Deposited Materials

[0094] A deposit containing a human NKAF II cDNA has been deposited withthe American Type Culture Collection, as noted above. Also as notedabove, the cDNA deposit is referred to herein as “the deposited clone”or as “the cDNA of the deposited clone.” The deposited clone wasdeposited with the American Type Culture Collection, 12301 Park LawnDrive, Rockville, Md. 20852, USA, on Mar. 6, 1996 and assigned ATCCDepositNo. 97465.

[0095] The deposited material is a pBluescript SK (-) plasmid(Stratagene, La Jolla, CA) that contains the full length NKAF II cDNA,referred to as “PF266” upon deposit.

[0096] The deposit has been made under the terms of the Budapest Treatyon the international recognition of the deposit of micro-organisms forpurposes of patent procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. § 112.

[0097] The sequence of the polynucleotides contained in the depositedmaterial, as well as the amino acid sequence of the polypeptide encodedthereby, are controlling in the event of any conflict with anydescription of sequences herein.

[0098] A license may be required to make, use or sell the depositedmaterials, and no such license is hereby granted.

[0099] Polypeptides

[0100] The present invention further relates to a human NKAF IIpolypeptide which has the deduced amino acid sequence of FIG. 1 (SEQ IDNO:2).

[0101] The invention also relates to fragments, analogs and derivativesof these polypeptides.

[0102] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide of FIG. 1 (SEQ ID NO:2) means a polypeptide whichretains essentially the same biological function or activity as suchpolypeptide. Thus, an analog includes a proprotein which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide.

[0103] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. Incertain preferred embodiments it is a recombinant polypeptide.

[0104] The fragment, derivative or analog of the polypeptide of FIG. 1(SEQ ID NO:2) may be (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code, or(ii) one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

[0105] Among the particularly preferred embodiments of the invention inthis regard are polypeptides having the amino acid sequence of NKAF IIset out in FIG. 1 (SEQ ID NO:2), variants, analogs, derivatives andfragments thereof, and variants, analogs and derivatives of thefragments. Alternatively, particularly preferred embodiments of theinvention in this regard are polypeptides having the amino acid sequenceof the NKAF II of the cDNA in the deposited clone, variants, analogs,derivatives and fragments thereof, and variants, analogs and derivativesof the fragments.

[0106] Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

[0107] Further particularly preferred in this regard are variants,analogs, derivatives and fragments, and variants, analogs andderivatives of the fragments, having the amino acid sequence of the NKAFII polypeptide of FIG. 1 (SEQ ID NO:2) in which several, a few, 5 to 10,1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deletedor added, in any combination. Especially preferred among these aresilent substitutions, additions and deletions, which do not alter theproperties and activities of the NKAF II. Also especially preferred inthis regard are conservative substitutions. Most highly preferred arepolypeptides having the amino acid sequence of FIG. 1 (SEQ ID NO:2)without substitutions.

[0108] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity. The polypeptides of the present invention also include thepolypeptide of SEQ ID NO:2 (in particular the mature polypeptide) aswell as polypeptides which have at least 70% similarity (preferably atleast 70% identity) to the polypeptide of SEQ ID NO:2 and morepreferably at least 90% similarity (more preferably at least 90%identity) to the polypeptide of SEQ ID NO:2 and still more preferably atleast 95% similarity (still more preferably at least 95% identity) tothe polypeptide of SEQ ID NO:2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0109] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0110] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.

[0111] Fragments or portions of the polynucleotides of the presentinvention may be used to synthesize full-length polynucleotides of thepresent invention.

[0112] Fragments

[0113] Also among preferred embodiments of this aspect of the presentinvention are polypeptides comprising fragments of NKAF II, mostparticularly fragments of the NKAF II having the amino acid set out inFIG. 1 (SEQ ID NO:2), and fragments of variants and derivatives of theNKAF II of FIG. 1 (SEQ ID NO:2).

[0114] In this regard a fragment is a polypeptide having an amino acidsequence that entirely is the same as part but not all of the amino acidsequence of the aforementioned NKAF II polypeptides and variants orderivatives thereof.

[0115] Such fragments may be “free-standing,” i.e., not part of or fusedto other amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of a NKAF IIpolypeptide of the present comprised within a precursor polypeptidedesigned for expression in a host and having heterologous pre andpro-polypeptide regions fused to the amino terminus of the NKAF IIfragment and an additional region fused to the carboxyl terminus of thefragment. Therefore, fragments in one aspect of the meaning intendedherein, refers to the portion or portions of a fusion polypeptide orfusion protein derived from NKAF II.

[0116] As representative examples of polypeptide fragments of theinvention, there may be mentioned those which have from about 21 toabout 225 amino acids.

[0117] In this context about includes the particularly recited range andranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acidat either extreme or at both extremes. For instance, about 225 aminoacids in this context means a polypeptide fragment of 225 plus or minusseveral, a few, 5, 4, 3, 2 or 1 amino acids to 225 plus or minus severala few, 5, 4, 3, 2 or 1 amino acid residues, i.e., ranges as broad as 21minus several amino acids to 225 plus several amino acids to as narrowas 21 plus several amino acids to 225 minus several amino acids.

[0118] Highly preferred in this regard are the recited ranges plus orminus as many as 5 amino acids at either or at both extremes.Particularly highly preferred are the recited ranges plus or minus asmany as 3 amino acids at either or at both the recited extremes.Especially particularly highly preferred are ranges plus or minus 1amino acid at either or at both extremes or the recited ranges with noadditions or deletions. Most highly preferred of all in this regard arefragments from about 21 to about 225 amino acids.

[0119] Among especially preferred fragments of the invention aretruncation mutants of NKAF II. Truncation mutants include NKAF IIpolypeptides having the amino acid sequence of FIG. 1 (SEQ ID NO:2), orof variants or derivatives thereof, except for deletion of a continuousseries of residues (that is, a continuous region, part or portion) thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus or, as in double truncation mutants,deletion of two continuous series of residues, one including the aminoterminus and one including the carboxyl terminus. Fragments having thesize ranges set out about also are preferred embodiments of truncationfragments, which are especially preferred among fragments generally.

[0120] Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of NKAF II.Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet-forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of NKAF II.

[0121] Certain preferred regions in these regards include, but are notlimited to, regions of the aforementioned types identified by analysisof the amino acid sequence set out in FIG. 1 (SEQ ID NO: 2). Suchpreferred regions include Gamier-Robson alpha-regions, beta-regions,turn-regions and coil-regions, Chou-Fasman alpha-regions, beta-regionsand turn-regions, Kyte-Doolittle hydrophilic regions and hydrophilicregions, Eisenberg alpha and beta amphipathic regions, Karplus-Schulzflexible regions, Emini surface-forming regions and Jameson-Wolf highantigenic index regions.

[0122] Among highly preferred fragments in this regard are those thatcomprise regions of NKAF II that combine several structural features,such as several of the features set out above.

[0123] In this regard, the regions defined by the residues about 19 to225, 20 to 225 and 21 to 225 of FIG. 1 (2 to 208, 3 to 208, and 4 to 208in SEQ ID NO:2, respectively), which all are characterized by amino acidcompositions highly characteristic of turn-regions, hydrophilic regions,flexible-regions, surface-forming regions, and high antigenicindex-regions, are especially highly preferred regions. Such regions maybe comprised within a larger polypeptide or may be by themselves apreferred fragment of the present invention, as discussed above. It willbe appreciated that the term “about” as used in this paragraph has themeaning set out above regarding fragments in general.

[0124] Further preferred regions are those that mediate activities ofNKAF II. Most highly preferred in this regard are fragments that have achemical, biological or other activity of NKAF II, including those witha similar activity or an improved activity, or with a decreasedundesirable activity. Highly preferred in this regard are fragments thatcontain regions that are homologs in sequence, or in position, or inboth sequence and to active regions of related polypeptides, such as therelated polypeptides set out in FIG. 2 (SEQ ID NO:l 1), which includehuman eosinophil granule major basic protein. Amongparticularlypreferred fragments in these regards are truncation mutants,as discussed above.

[0125] It will be appreciated that the invention also relates to, amongothers, polynucleotides encoding the aforementioned fragments,polynucleotides that hybridize to polynucleotides encoding thefragments, particularly those that hybridize under stringent conditions,and polynucleotides, such as PCR primers, for amplifying polynucleotidesthat encode the fragments. In these regards, preferred polynucleotidesare those that correspondent to the preferred fragments, as discussedabove.

[0126] Vectors, Host Cells and Expression

[0127] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0128] Host cells can be genetically engineered to incorporatepolynucleotides and express polypeptides of the present invention. Forinstance, polynucleotides may be introduced into host cells using wellknown techniques of infection, transduction, transfection, transvectionand transformation. The polynucleotides may be introduced alone or withother polynucleotides. Such other polynucleotides may be introducedindependently, co-introduced or introduced joined to thepolynucleotidesofthe invention.

[0129] Thus, for instance, polynucleotides of the invention may betransfected into host cells with another, separate, polynucleotideencoding a selectable marker, using standard techniques forco-transfection and selection in, for instance, mammalian cells. In thiscase the polynucleotides generally will be stably incorporated into thehost cell genome.

[0130] Alternatively, the polynucleotides may be joined to a vectorcontaining a selectable marker for propagation in a host. The vectorconstruct may be introduced into host cells by the aforementionedtechniques. Generally, a plasmid vector is introduced as DNA in aprecipitate, such as a calcium phosphate precipitate, or in a complexwith a charged lipid. Electroporation also may be used to introducepolynucleotides into a host. If the vector is a virus, it may bepackaged in vitro or introduced into a packaging cell and the packagedvirus may be transduced into cells. A wide variety of techniquessuitable for making polynucleotides and for introducing polynucleotidesinto cells in accordance with this aspect of the invention are wellknown and routine to those of skill in the art. Such techniques arereviewed at length in Sambrook et al. cited above, which is illustrativeof the many laboratory manuals that detail these techniques.

[0131] In accordance with this aspect of the invention the vector maybe, for example, a plasmid vector, a single or double-stranded phagevector, a single or double-stranded RNA or DNA viral vector. Suchvectors may be introduced into cells as polynucleotides, preferably DNA,by well known techniques for introducing DNA and RNA into cells. Thevectors, in the case of phage and viral vectors also may be andpreferably are introduced into cells as packaged or encapsidated virusby well known techniques for infection and transduction. Viral vectorsmay be replication competent or replication defective. In the lattercase viral propagation generally will occur only in complementing hostcells.

[0132] Preferred among vectors, in certain respects, are those forexpression of polynucleotides and polypeptides of the present invention.Generally, such vectors comprise cis-acting control regions effectivefor expression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

[0133] In certain preferred embodiments in this regard, the vectorsprovide for specific expression. Such specific expression may beinducible expression or expression only in certain types of cells orboth inducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

[0134] The engineered host cells can be cultured in conventionalnutrient media, which may be modified as appropriate for, inter alia,activating promoters, selecting transformants or amplifying genes.Culture conditions, such as temperature, pH and the like, previouslyused with the host cell selected for expression generally will besuitable for expression of polypeptides of the present invention as willbe apparent to those of skill in the art.

[0135] A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors e.g., vectors derived from bacterial plasmids,from bacteriophage, from yeast episomes, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids, all may be used for expression inaccordance with this aspect of the present invention. Generally, anyvector suitable to maintain, propagate or express polynucleotides toexpress a polypeptide in a host may be used for expression in thisregard.

[0136] The appropriate DNA sequence may be inserted into the vector byany of a variety of well-known and routine techniques. In general, a DNAsequence for expression is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. Procedures for restriction and ligation that can be usedto this end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseskill, are set forth in great detail in Sambrook et al. cited elsewhereherein.

[0137] The DNA sequence in the expression vector is operatively linkedto appropriate expression control sequence(s), including, for instance,a promoter to direct mRNA transcription. Representatives of suchpromoters include the phage lambda PL promoter, the E. coli lac, trp andtac promoters, the SV40 early and late promoters and promoters ofretroviral LTRs, to name just a few of the well-known promoters. It willbe understood that numerous promoters not mentioned are suitable for usein this aspect of the invention are well known and readily may beemployed by those of skill in the manner illustrated by the discussionand the examples herein.

[0138] In general, expression constructs will contain sites fortranscription initiation and termination, and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will include atranslation initiating AUG at the beginning and a termination codonappropriately positioned at the end of the polypeptide to be translated.

[0139] In addition, the constructs may contain control regions thatregulate as well as engender expression. Generally, in accordance withmany commonly practiced procedures, such regions will operate bycontrolling transcription, such as repressor binding sites andenhancers, among others.

[0140] Vectors for propagation and expression generally will includeselectable markers. Such markers also may be suitable for amplificationor the vectors may contain additional markers for this purpose. In thisregard, the expression vectors preferably contain one or more selectablemarker genes to provide a phenotypic trait for selection of transformedhost cells. Preferred markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture, and tetracycline,theomycin, kanamycin or ampicillin resistance genes for culturing E.coli and other bacteria.

[0141] The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Hosts for of agreat variety of expression constructs are well known, and those ofskill will be enabled by the present disclosure readily to select a hostfor expressing a polypeptides in accordance with this aspect of thepresent invention.

[0142] Various mammalian cell culture systems can be employed forexpression, as well. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblast, described in Gluzman etal., Cell 23: 175 (1981). Other cell lines capable of expressing acompatible vector include for example, the C 127, 3T3, CHO, HeLa, humankidney 293 and BHK cell lines.

[0143] More particularly, the present invention also includesrecombinant constructs, such as expression constructs, comprising one ormore of the sequences described above. The constructs comprise a vector,such as a plasmid or viral vector, into which such a sequence of theinvention has been inserted. The sequence may be inserted in a forwardor reverse orientation. In certain preferred embodiments in this regard,the construct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and there are many commercially available vectors suitable for use inthe present invention.

[0144] The following vectors, which are commercially available, areprovided by way of example. Among vectors preferred for use in bacteriaare pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors,Phagescript vectors, Bluescript vectors, pNH8A, pNH 1 6a, pNH 1 8A,pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 available from Pharmacia. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.These vectors are listed solely by way of illustration of the manycommercially available and well known vectors that are available tothose of skill in the art for use in accordance with this aspect of thepresent invention. It will be appreciated that any other plasmid orvector suitable for, for example, introduction, maintenance, propagationor expression of a polynucleotide or polypeptide of the invention in ahost may be used in this aspect of the invention.

[0145] Promoter regions can be selected from any desired gene usingvectors that contain a reporter transcription unit lacking a promoterregion, such as a chloramphenicol acetyl transferase (“cat”)transcription unit, downstream of restriction site or sites forintroducing a candidate promoter fragment; i.e., a fragment that maycontain a promoter. As is well known, introduction into the vector of apromoter-containing fragment at the restriction site upstream of the catgene engenders production of CAT activity, which can be detected bystandard CAT assays. Vectors suitable to this end are well known andreadily available. Two such vectors are pKK232-8 and pCM7. Thus,promoters for expression of polynucleotides of the present inventioninclude not only well known and readily available promoters, but alsopromoters that readily may be obtained by the foregoing technique, usinga reporter gene.

[0146] Among known bacterial promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lacd and lacZ promoters, the T3 and T7promoters, the T5 tac promoter, the lambda PR, PL promoters and the trppromoter. Among known eukaryotic promoters suitable in this regard arethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (“RSV”), and metallothioneinpromoters,such as the mouse metallothionein-I promoter.

[0147] Selection of appropriate vectors and promoters for expression ina host cell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

[0148] Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector.

[0149] The present invention also relates to host cells containing theabove-described constructs discussed above. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell.

[0150] Constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can besyntheticallyproducedby conventional peptide synthesizers.

[0151] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989).

[0152] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from I0 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0153] Polynucleotides of the invention, encoding the heterologousstructural sequence of a polypeptide of the invention generally will beinserted into the vector using standard techniques so that it isoperably linked to the promoter for expression. The polynucleotide willbe positioned so that the transcription start site is locatedappropriately 5′ to a ribosome binding site. The ribosome binding sitewill be 5′ to the AUG that initiates translation of the polypeptide tobe expressed. Generally, there will be no other open reading frames thatbegin with an initiation codon, usually AUG, and lie between theribosome binding site and the initiating AUG. Also, generally, therewill be a translation stop codon at the end of the polypeptide and therewill be a polyadenylation signal and a transcription termination signalappropriately disposed at the 3′ end of the transcribed region.

[0154] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

[0155] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals but alsoadditional heterologous functional regions. Thus, for instance, a regionof additional amino acids, particularly charged amino acids, may beadded to the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art.

[0156] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, where the selectedpromoter is inducible it is induced by appropriate means (e.g.,temperature shift or exposure to chemical inducer) and cells arecultured for an additional period.

[0157] Cells typically then are harvested by centrifugation, disruptedby physical or chemical means, and the resulting crude extract retainedfor further purification.

[0158] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0159] The NKAF II polypeptide can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Well known techniques for refolding protein may be employed toregenerate active conformation when the polypeptide is denatured duringisolation and or purification.

[0160] Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediatedprocesses.

[0161] NKAF II polynucleotides and polypeptides may be used inaccordance with the present invention for a variety of applications,particularly those that make use of the chemical and biologicalproperties of NKAF II. Among these are applications in prevention ofneoplasia. Additional applications relate to diagnosis and to treatmentof disorders of cells, tissues and organisms. These aspects of theinvention are illustrated further by the following discussion.

[0162] Polynucleotide Assays

[0163] This invention is also related to the use of the NKAF IIpolynucleotides to detect complementary polynucleotides such as, forexample, as a diagnostic reagent. Detection of a mutated form of NKAF IIassociated with a dysfunction will provide a diagnostic tool that canadd or define a diagnosis of a disease or susceptibility to a diseasewhich results from under-expression over-expressionor altered expressionof NKAF II, such as, for example, eosinophilia or bronchial asthma.

[0164] Individuals carrying mutations in the human NKAF II gene may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR prior to analysis. PCR (Saiki et al., Nature, 324: 163-166(1986)). RNA or cDNA may also be used in the same ways. As an example,PCR primers complementary to the nucleic acid encoding NKAF II can beused to identify and analyze NKAF II expression and mutations. Forexample, deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Pointmutations can be identified by hybridizing amplified DNA to radiolabeledNKAF II RNA or alternatively, radiolabeled NKAF II antisense DNAsequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

[0165] Sequence differences between a reference gene and genes havingmutations also may be revealed by direct DNA sequencing. In addition,cloned DNA segments may be employed as probes to detect specific DNAsegments. The sensitivity of such methods can be greatly enhanced byappropriate use of PCR or another amplification method. For example, asequencing primer is used with double-stranded PCR product or asingle-stranded template molecule generated by a modified PCR. Thesequence determination is performed by conventional procedures withradiolabeled nucleotide or by automatic sequencing procedures withfluorescent-tags.

[0166] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230: 1242 (1985)).

[0167] Sequence changes at specific locations also may be revealed bynuclease protection assays, such as RNase and SI protection or thechemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci.,USA, 85: 4397-4401 (1985)).

[0168] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms (“RFLP”) and Southern blottingof genomic DNA.

[0169] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations also can be detected by in situ analysis.

[0170] Chromosome Assays

[0171] The sequences of the present invention are also valuable forchromosome identification.

[0172] The sequence is specifically targeted to and can hybridize with aparticular location on an individual human chromosome. Moreover, thereis a current need for identifyingparticular sites on the chromosome. Fewchromosome marking reagents based on actual sequence data (repeatpolymorphisms) are presently available for marking chromosomal location.The mapping of DNAs to chromosomes according to the present invention isan important first step in correlating those sequences with genesassociated with disease.

[0173] In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a NKAF II gene. This can beaccomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA the is usedfor in situ chromosome mapping using well known techniques for thispurpose. Typically, in accordance with routine procedures for chromosomemapping, some trial and error may be necessary to identify a genomicprobe that gives a good in situ hybridization signal.

[0174] In some cases, in addition, sequences can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp) from thecDNA. Computer analysis of the 3′ untranslated region of the gene isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the primer will yield an amplifiedfragment.

[0175] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0176] Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press,New York (1988).

[0177] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelatedwith genetic map data. Such data are found, for example, in V.McKusick, MENDELIAN INHERITANCE IN MAN, available on line through JohnsHopkins University, Welch Medical Library. The relationship betweengenes and diseases that have been mapped to the same chromosomal regionare then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0178] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0179] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes I megabase mapping resolution and onegene per 20 kb).

[0180] Polypeptide Assays

[0181] The present invention also relates to a diagnostic assays such asquantitative and diagnostic assays for detecting levels of NKAF IIprotein in cells and tissues, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting over-expression of NKAF II proteincompared to normal control tissue samples may be used to detect thepresence of eosinophilia, for example. Assay techniques that can be usedto determine levels of a protein, such as an NKAF II protein of thepresent invention, in a sample derived from a host are well-known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.Among these ELISAs frequently are preferred. An ELISA assay initiallycomprises preparing an antibody specific to NKAF II, preferably amonoclonal antibody. In addition a reporter antibody generally isprepared which binds to the monoclonal antibody. The reporter antibodyis attached a detectable reagent such as radioactive, fluorescent orenzymatic reagent, in this example horseradish peroxidase enzyme.

[0182] To carry out an ELISA a sample is removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein such as bovineserum albumin. Next, the monoclonal antibody is incubated in the dishduring which time the monoclonal antibodies attach to any NKAF IIproteins attached to the polystyrene dish. Unbound monoclonal antibodyis washed out with buffer. The reporter antibody linked to horseradishperoxidase is placed in the dish resulting in binding of the reporterantibody to any monoclonal antibody bound to NKAF II. Unattachedreporter antibody is then washed out. Reagents for peroxidase activity,including a colorimetric substrate are then added to the dish.Immobilized peroxidase, linked to NKAF II through the primary andsecondary antibodies, produces a colored reaction product. The amount ofcolor developed in a given time period indicates the amount of NKAF IIprotein present in the sample. Quantitative results typically areobtained by reference to a standard curve.

[0183] A competition assay may be employed wherein antibodies specificto NKAF II attached to a solid support and labeled NKAF II and a samplederived from the host are passed over the solid support and the amountof label detected attached to the solid support can be correlated to aquantity of NKAF II in the sample.

[0184] Antibodies

[0185] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0186] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0187] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler, G. and Milstein,C., Nature 256: 495-497 (1975), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4: 72 (1983) andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985).

[0188] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice, or other organisms such as other mammals, may beused to express humanized antibodies to immunogenic polypeptide productsof this invention.

[0189] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or purify the polypeptide ofthe present invention by attachment of the antibody to a solid supportfor isolation and/or purification by affinity chromatography.

[0190] Thus, among others, NKAF II may be employed to inhibit the growthof certain tumor cell lines, and other neoplasias due to the cytotoxiceffect of NKAF II.

[0191] The polypeptide of the present invention may also be employed toprevent inflammation, to treat parasitic infection due to its cytotoxiceffect on certain parasites, to augment the effects of natural killerprotein to treat neoplasias such as tumors and cancers, to preventinflammation, to treat parasitic infection, to regulate hemaetopoesis,to prevent damage from superoxide radicals in the body, for example, toprevent tissue injury and aging and to enhance an immunologicalresponse.

[0192] NKAF II Binding Molecules and Assays

[0193] This invention also provides a method for identification ofmolecules, such as receptor molecules, that bind NKAF II. Genes encodingproteins that bind NKAF II, such as receptor proteins, can be identifiedby numerous methods known to those of skill in the art, for example,ligand panning and FACS sorting. Such methods are described in manylaboratory manuals such as, for instance, Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

[0194] For instance, expression cloning may be employed for thispurpose. To this end polyadenylated RNA is prepared from a cellresponsive to NKAF II, a cDNA library is created from this RNA, thelibrary is divided into pools and the pools are transfected individuallyinto cells that are not responsive to NKAF II. The transfected cellsthen are exposed to labeled NKAF II. (NKAF II can be labeled by avariety of well-known techniques including standard methods ofradio-iodinationor inclusion of a recognition site for a site-specificprotein kinase.) Following exposure, the cells are fixed and binding ofNKAF II is determined. These procedures conveniently are carried out onglass slides.

[0195] Pools are identified of cDNA that produced NKAF II-binding cells.Sub-pools are prepared from these positives, transfected into host cellsand screened as described above. Using an iterative sub-pooling andre-screening process, plasmids containing one or more single clones thatencode the putative binding molecule, such as a receptor molecule, canbe isolated and the clones are sequenced.

[0196] Alternatively a labeled ligand can be photoaffinity linked to acell extract, such as a membrane or a membrane extract, prepared fromcells that express a molecule that it binds, such as a receptormolecule. Cross-linked material is resolved by polyacrylamide gelelectrophoresis (“PAGE”) and exposed to X-ray film. The labeled complexcontaining the ligand-receptor can be excised, resolved into peptidefragments, and subjected to protein microsequencing. The amino acidsequence obtained from microsequencing can be used to design unique ordegenerate oligonucleotide probes to screen cDNA libraries to identifygenes encoding the putative receptor molecule.

[0197] Polypeptides of the invention also can be used to assess NKAF IIbinding capacity of NKAF II binding molecules, such as receptormolecules, in cells or in cell-free preparations.

[0198] Agonists and Antagonists—Assays and Molecules

[0199] The invention also provides a method of screening compounds toidentify those which enhance or block the action of NKAF II on cells,such as its interaction with NKAF II-binding molecules such as receptormolecules. An agonist is a compound which increases the naturalbiological functions of NKAF II or which functions in a manner similarto NKAF II, while antagonists decrease or eliminate such functions.

[0200] For example, a cellular compartment, such as a membrane or apreparation thereof, such as a membrane-preparation, may be preparedfrom a cell that expresses a molecule that binds NKAF II, such as amolecule of a signaling or regulatory pathway modulated by NKAF II. Thepreparation is incubated with labeled NKAF II in the absence or thepresence of a candidate molecule which may be a NKAF II agonist orantagonist. The ability of the candidate molecule to bind the bindingmolecule is reflected in decreased binding of the labeled ligand.Molecules which bind gratuitously, i.e., without inducing the effects ofNKAF II on binding the NKAF II binding molecule, are most likely to begood antagonists. Molecules that bind well and elicit effects that arethe same as or closely related to NKAF II, are good agonists.

[0201] NKAF II-like effects of potential agonists and antagonists may bymeasured, for instance, by determining activity of a second messengersystem following interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that of NKAFII or molecules that elicit the same effects as NKAF II. Secondmessenger systems that may be useful in this regard include but are notlimited to AMP guanylate cyclase, ion channel or phosphoinositidehydrolysis second messenger systems.

[0202] Another example of an assay for NKAF II antagonists is acompetitive assay that combines NKAF II and a potential antagonist withmembrane-bound NKAF II receptor molecules or recombinant NKAF IIreceptor molecules under appropriate conditions for a competitiveinhibition assay. NKAF II can be labeled, such as by radioactivity, suchthat the number of NKAF II molecules bound to a receptor molecule can bedetermined accurately to assess the effectiveness of the potentialantagonist.

[0203] Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducing NKAFII-induced activities, thereby preventing the action of NKAF II byexcluding NKAF II from binding.

[0204] Other potential antagonists include antisense molecules.Antisense technology can be used to control gene expression throughantisense DNA or RNA or through triple-helix formation. Antisensetechniques are discussed, for example, in—Okano, J. Neurochem. 56: 560(1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENEEXPRESSION, CRC Press, Boca Raton, FL (1988). Triple helix formation isdiscussed in, for instance Lee et al., Nucleic Acids Research 6: 3073(1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,Science 251: 1360 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA. For example, the 5′ codingportion of a polynucleotide that encodes the mature polypeptide of thepresent invention may be used to design an antisense RNA oligonucleotideof from about 10 to 40 base pairs in length. A DNA oligonucleotide isdesigned to be complementary to a region of the gene (or promotor)involved in transcription thereby preventing transcription and theproduction of NKAF II. The antisense RNA oligonucleotide hybridizes tothe mRNA in vivo and blocks translation of the mRNA molecule into NKAFII polypeptide. The oligonucleotides described above can also bedelivered to cells such that the antisense RNA or DNA may be expressedin vivo to inhibit production of NKAF II.

[0205] The antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

[0206] The antagonists may be employed for instance to treat and/orprevent the action of such polypeptide, for example, to prevent allergicinflammation, hypersensitivity, bronchial asthma, eosinophilia, chronicurticaria, atopic dermatitis, Kimura's disease, and bone marrowtransplant rejection.

[0207] Compositions

[0208] The invention also relates to compositions comprising thepolynucleotide or the polypeptides discussed above or the agonists orantagonists. Thus, the polypeptides of the present invention may beemployed in combination with a non-sterile or sterile carrier orcarriers for use with cells, tissues or organisms, such as apharmaceutical carrier suitable for administration to a subject. Suchcompositions comprise, for instance, a media additive or atherapeutically effective amount of a polypeptide of the invention and apharmaceutically acceptable carrier or excipient. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,water, glycerol, ethanol and combinations thereof. The formulationshould suit the mode of administration.

[0209] Kits

[0210] The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

[0211] Administration

[0212] Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0213] The pharmaceutical compositions may be administered in anyeffective, convenient manner including, for instance, administration bytopical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes amongothers.

[0214] The pharmaceutical compositions generally are administered in anamount effective for treatment or prophylaxis of a specific indicationor indications. In general, the compositions are administered in anamount of at least about 10 mg/kg body weight. In most cases they willbe administered in an amount not in excess of about 8 mg/kg body weightper day. Preferably, in most cases, dose is from about 10 mg/kg to about1 mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

[0215] Gene Therapy

[0216] The NKAF II polynucleotides, polypeptides, agonists andantagonists that are polypeptides may be employed in accordance with thepresent invention by expression of such polypeptides in vivo, intreatment modalities often referred to as “gene therapy.”

[0217] Thus, for example, cells from a patient may be engineered with apolynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo,and the engineered cells then can be provided to a patient to be treatedwith the polypeptide. For example, cells may be engineered ex vivo bythe use of a retroviral plasmid vector containing RNA encoding apolypeptide of the present invention. Such methods are well-known in theart and their use in the present invention will be apparent from theteachings herein.

[0218] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by procedures known in the art. For example, apolynucleotide of the invention may be engineered for expression in areplication defective retroviral vector, as discussed above. Theretroviral expression construct then may be isolated and introduced intoa packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

[0219] Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, MoloneyMurine Leukemia Virus, spleen necrosis virus, retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon apeleukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0220] Such vectors well include one or more promoters for expressingthe polypeptide. Suitable promoters which may be employed include, butare not limited to, the retroviral LTR;

[0221] the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller et al., Biotechniques 7: 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, RNA polymeraseIII, and β-actin promoters). Other viral promoters which may be employedinclude, but are not limited to, adenovirus promoters, thymidine kinase(TK) promoters, and B19 parvovirus promoters. The selection of asuitable promoter will be apparent to those skilled in the art from theteachings contained herein.

[0222] The nucleic acid sequence encoding the polypeptide of the presentinvention will be placed under the control of a suitable promoter.Suitable promoters which may be employed include, but are not limitedto, adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0223] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, Y-2, Y-AM, PA12, T19-14X, VT-19-17-H2, YCRE, YCRIP, GP+E-86,GP+envAml 2, and DAN cell lines as described in Miller, A., Human GeneTherapy 1: 5-14 (1990). The vector may be transduced into the packagingcells through any means known in the art. Such means include, but arenot limited to, electroporation, the use of liposomes, and CaPO4precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministeredto a host.

[0224] The producer cell line will generate infectious retroviral vectorparticles, which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

EXAMPLES

[0225] The present invention is further described by the followingexamples. The examples are provided solely to illustrate the inventionby reference to specific embodiments. These exemplification's, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

[0226] Certain terms used herein are explained in the foregoingglossary.

[0227] All examples were carried out using standard techniques, whichare well known and routine to those of skill in the art, except whereotherwise described in detail. Routine molecular biology techniques ofthe following examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), herein referred to as “Sambrook.”

[0228] All parts or amounts set out in the following examples are byweight, unless otherwise specified.

[0229] Unless otherwise stated size separation of fragments in theexamples below was carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis (“PAGE”) in Sambrook and numerousother references such as, for instance, by Goeddel et al., Nucleic AcidsRes. 8: 4057 (1980).

[0230] Unless described otherwise, ligations were accomplished usingstandard buffers, incubation temperatures and times, approximatelyequimolar amounts of the DNA fragments to be ligated and approximately10 units of T4 DNA ligase (“ligase”) per 0.5 mg of DNA.

Example 1

[0231] Expression and Purification of Mature Human NKAF II UsingBacteria

[0232] The DNA sequence encoding human NKAF II in the depositedpolynucleotide was amplified using PCR oligonucleotide primers specificto the amino acid carboxyl terminal sequence of the human NKAF IIprotein and to vector sequences 3′ to the gene. Additional nucleotidescontaining restriction sites to facilitate cloning were added to the 5′and 3′ sequences respectively.

[0233] The 5′ oligonucleotide primer had the sequence 5′ CGC +E,unsCCATGG AGA ATG ATG CCC CCC AT 3′ (SEQ ID NO:3) containing the underlinedNco I restriction site, which encodes a start AUG, followed by 17nucleotides of the human NKAF II coding sequence set out in FIG. 1 (SEQID NO:1) beginning with the first base of the first mature codon aspredicted by the computer program PSORT. The molecule encodes aparticularly preferred fragment of the mature NKAF II polypeptide havingthe amino acid sequence shown as amino acid residues 3 to 208 in SEQ IDNO:2. The 3′ primer had the sequence 5′ CGC +E,uns AAGCTT CTC CGT GCCGCT GGC TTA 3′ (SEQ ID NO:4) containing the underlined Hind IIIrestriction site followed by nucleotides complementary to the last 18NKAF II non-coding sequence next to stop codon set out in FIG. 1 (SEQ IDNO:1), including the stop codon.

[0234] The restrictions sites were convenient to restriction enzymesites in the bacterial expression vectors pQE-60, which were used forbacterial expression in these examples. (Qiagen, Inc. Chatsworth, CA).pQE-60 encodes ampicillin antibiotic resistance (“Ampr”) and contains abacterial origin of replication (“ori”), an IPTG inducible promoter, aribosome binding site (“RBS”), a 6-His tag and restriction enzyme sites.

[0235] The amplified human NKAF II DNA and the vector pQE-60 both weredigested with Nco I and Hind III and the digested DNAs then were ligatedtogether. Insertion of the NKAF II DNA into the restricted vector placedthe NKAF II coding region downstream of and operably linked to thevector's IPTG-inducible promoter and in-frame with an initiating AUGappropriately positioned for translation of NKAF II.

[0236] The ligation mixture was transformed into competent E. coli cellsusing standard procedures. Such procedures are described in Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kanr”), wasused in carrying out the illustrative example described here. Thisstrain, which is only one of many that are suitable for expressing NKAFII, is available commercially from Qiagen.

[0237] Transformants were identified by their ability to grow on LBplates in the presence of ampicillin. Plasmid DNA was isolated fromresistant colonies and the identity of the cloned DNA was confirmed byrestriction analysis.

[0238] Clones containing the desired constructs were grown overnight(“O/N”) in liquid culture in LB media supplemented with both ampicillin(100 ug/ml) and kanamycin (25 ug/ml).

[0239] The O/N culture was used to inoculate a large culture, at adilution of approximately 1:100 to 1:250. The cells were grown to anoptical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) was then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the laci repressor. Cellssubsequently were incubated further for 3 to 4 hours. Cells then wereharvested by centrifugation and disrupted, by standard methods.Inclusion bodies were purified from the disrupted cells using routinecollection techniques, and protein was solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein was passed over a PD-10 column in 2×phosphate buffered saline(“PBS”), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein was purified by a further step ofchromatography to remove endotoxin. Then, it was sterile filtered. Thesterile filtered protein preparation was stored in 2X PBS at aconcentration of 95 micrograms per mL.

[0240] Analysis of the preparation by standard methods of polyacrylamidegel electrophoresis revealed that the preparation contained about 95%monomer NKAF II having the expected molecular weight of, approximately,23.3 kDa.

Example 2

[0241] Cloning and Expression of Human NKAF II in a BaculovirusExpression System

[0242] The cDNA sequence encoding the full length human NKAF II protein,in the deposited clone is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene:

[0243] The 5′ primer has the sequence 5′ CGC +E,uns GGATCC GCC ATC ATGCAA CGC CTC TTG 3′ (SEQ ID NO:5) containing the underlined Bam HIrestriction enzyme site followed by 15 bases of the sequence of NKAF IIof FIG. 1 (SEQ ID NO:1). Inserted into an expression vector, asdescribed below, the 5′ end of the amplified fragment encoding humanNKAF II provides an efficient signal peptide. An efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196: 947-950 (1987) is appropriately located in thevector portion of the construct.

[0244] The 3′ primer has the sequence 5° CGC +E,uns GGT ACC CTC CGT GCCGCT GGC TTA 3′ (SEQ ID NO:6) containing the underlined Asp718restriction followed by nucleotides complementary to 18 nucleotides ofNKAF II non-coding sequence next to stop codon.

[0245] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with BamHI and Asp7l8 and againis purified on a 1% agarose gel. This fragment is designated herein F2.

[0246] The vector pA2 is used to express the NKAF II protein in thebaculovirus expression system, using standard methods, such as thosedescribed in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORSAND INSECT CELL CULTURE PROCEDURES, Texas Agricultural ExperimentalStation Bulletin No. 1555 (1987). This expression vector contains thestrong polyhedrin promoter of the Autographa califomica nuclearpolyhedrosis virus (AcMNPV) followed by convenient restriction sites.The signal peptide of AcMNPV gp67, including the N-terminal methionine,is located just upstream of a BamHI site. The polyadenylation site ofthe simian virus 40 (“SV40 ”) is used for efficient polyadenylation. Foran easy selection of recombinant virus the beta-galactosidasegene fromE.coli is inserted in the same orientation as the polyhedrin promoterand is followed by the polyadenylation signal of the polyhedrin gene.The polyhedrin sequences are flanked at both sides by viral sequencesfor cell-mediated homologous recombination with wild-type viral DNA togenerate viable virus that express the cloned polynucleotide.

[0247] Many other baculovirus vectors could be used in place of pA2,such as pAc373, pVL941 and pAcIM1 provided, as those of skill readilywill appreciate, that construction provides appropriately locatedsignals for transcription, translation, trafficking and the like, suchas an in-frame AUG and a signal peptide, as required. Such vectors aredescribed in Luckow et al., Virology 170: 31-39, among others.

[0248] The plasmid is digested with the restriction enzymes and Bam HIand Asp718 and then is dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., LaJolla, Calif.). This vector DNA isdesignated herein “V2”.

[0249] Fragment F2 and the dephosphorylated plasmid V2 are ligatedtogether with T4 DNA ligase. E.coli HB101 cells are transformed withligation mix and spread on culture plates. Bacteria are identified thatcontain the plasmid with the human NKAF II gene by digesting DNA fromindividual colonies using Bam HI and Asp7l8 and then analyzing thedigestion product by gel electrophoresis. The sequence of the clonedfragment is confirmed by DNA sequencing. This plasmid is designatedherein pBacNKAF II.

[0250] 5 mg of the plasmid pBacNKAF II is co-transfected with 1.0 mg ofa commercially available linearized baculovirus DNA (“BaculoGoldÔbaculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). 1 mg of BaculoGoldÔ virus DNA and 5 mg of the plasmidpBacNKAF II are mixed in a sterile well of a microtiter plate containing50 ml of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 ml Lipofectin plus 90 ml Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature.

[0251] Then the transfectionmixture is added drop-wise to Sf9 insectcells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 mlGrace's medium without serum. The plate is rocked back and forth to mixthe newly added solution. The plate is then incubated for 5 hours at 27°C. After 5 hours the transfection solution is removed from the plate and1 ml of Grace's insect medium supplemented with 10% fetal calf serum isadded. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

[0252] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, cited above. An agarosegel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirologydistributedby Life Technologies Inc.,Gaithersburg, page 9-10).

[0253] Four days after serial dilution, the virus is added to the cells.After appropriate incubation, blue stained plaques are picked with thetip of an Eppendorf pipette. The agar containing the recombinant virusesis then resuspended in an Eppendorf tube containing 200 ml of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. A clonecontaining properly inserted NKAF II is identified by DNA analysisincluding restriction mapping and sequencing. This is designated hereinas V-NKAF II.

[0254] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-NKAF II at a multiplicity of infection (“MOI”) of about 2(about 1 to about 3). Six hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Gaithersburg). 42 hours later, 5 mCi of35S-methionine and 5 mCi 35S cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

[0255] The N-terminal sequence of the NKAF II polypeptide producedaccording to the above method was determined to begin with the aminoacid residue I as shown in SEQ ID NO:2.

Example 3

[0256] Expression of NKAF II in COS Cells

[0257] The expression plasmid, NKAF II HA, is made by cloning a cDNAencoding NKAF II into the expression vector pcDNAI/Amp (which can beobtained from Invitrogen, Inc.).

[0258] The expression vector pcDNAI/amp contains: (1) an E.coli originof replication effective for propagation in E. coli and otherprokaryotic cell; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron, and a polyadenylation signal arranged so that a cDNAconveniently can be placed under expression control of the CMV promoterand operably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker.

[0259] A DNA fragment encoding the entire NKAF II precursor and a HA tagfused in frame to its 3′ end is cloned into the polylinker region of thevector so that recombinant protein expression is directed by the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein described by Wilson et al., Cell 37: 767(1984). The fusion of the HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

[0260] The plasmid construction strategy is as follows.

[0261] The NKAF II cDNA of the deposit clone is amplified using primersthat contained convenient restriction sites, much as described aboveregarding the construction of expression vectors for expression of NKAFII in E. coli and S. furgiperda.

[0262] To facilitate detection, purification and characterization of theexpressed NKAF II, one of the primers contains a heamaglutinin tag (“HAtag”) as described above.

[0263] Suitable primers include that following, which are used in thisexample.

[0264] The 5′ primer 5′ CGC +E,uns GGATCC GCC ATC ATG CAA CGC CTC TTG 3′(SEQ ID NO: 7) contains the underlined Bam HI site, an AUG start codonand codons thereafter.

[0265] The 3′ primer, containing the underlined Xba I site; 15 bp of 3′coding sequence (at the 3′ end); and hemagluttinin tag has the followingsequence, 5′ CGC +E,uns TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTAGAA GGA GCA GAC GAA 3′ (SEQ ID NO:8).

[0266] The PCR amplified DNA fragment and the vector, pcDNAI/Amp, aredigested with and then ligated. The ligation mixture is transformed intoE. coli strain SURE (available from Stratagene Cloning Systems, 11099North Torrey Pines Road, La Jolla, Calif. 92037) the transformed cultureis plated on ampicillin media plates which then are incubated to allowgrowth of ampicillin resistant colonies. Plasmid DNA is isolated fromresistant colonies and examined by restriction analysis and gel sizingfor the presence of the NKAF II-encoding fragment.

[0267] For expression of recombinant NKAF II, COS cells are transfectedwith an expression vector, as described above, using DEAE-DEXTRAN, asdescribed, for instance, in Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, Cold Spring Laboratory Press, Cold Spring Harbor, NewYork (1989).

[0268] Cells are incubated under conditions for expression of NKAF II bythe vector.

[0269] Expression of the NKAF II HA fusion protein is detected byradiolabelling and immunoprecipitation, using methods described in, forexample Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing 35S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containingRIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE gels and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 4

[0270] Tissue Distribution of NKAF II Expression

[0271] Northern blot analysis is carried out to examine the levels ofexpression of NKAF II in human tissues, using methods described by,among others, Sambrook et al, cited above. Total cellular RNA samplesare isolated with RNAzolÔ B system (Biotecx Laboratories, Inc. 6023South Loop East, Houston, Tex. 77033).

[0272] About 10 mg of Total RNA is isolated from tissue samples. The RNAis size resolved by electrophoresis through a 1% agarose gel understrongly denaturing conditions. RNA is blotted from the gel onto a nylonfilter, and the filter then is prepared for hybridization to adetectably labeled polynucleotide probe.

[0273] As a probe to detect mRNA that encodes NKAF II, the antisensestrand of the coding region of the cDNA insert in the deposited clone islabeled to a high specific activity. The cDNA is labeled by primerextension, using the Prime-It kit, available from Stratagene. Thereaction is carried out using 50 ng of the cDNA, following the standardreaction protocol as recommended by the supplier. The labeledpolynucleotide is purified away from other labeled reaction componentsby column chromatography using a Select-G-50 column, obtained from5-Prime-3-Prime, Inc. of 5603 Arapahoe Road, Boulder, Colo. 80303.

[0274] The labeled probe is hybridized to the filter, at a concentrationof 1,000,000 cpm/ml, in a small volume of 7% SDS, 0.5 M NaPO4, pH 7.4 at65° C., overnight.

[0275] Thereafter the probe solution is drained and the filter is washedtwice at room temperature and twice at 60° C. with 0.5×SSC, 0.1% SDS.The filter then is dried and exposed to film at −70° C. overnight withan intensifying screen.

[0276] Autoradiography shows that mRNA for NKAF II is abundant in fetalliver and bone marrow.

Example 5

[0277] Gene Therapeutic Expression of Human NKAF II

[0278] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature overnight. After 24 hours at room temperature, the flask isinverted—the chunks of tissue remain fixed to the bottom of theflask—and fresh media is added (e.g., Ham's F12 media, with 10% FBS,penicillin and streptomycin). The tissue is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerges. The monolayer istrypsinized and scaled into larger flasks.

[0279] A vector for gene therapy is digested with restriction enzymesfor cloning a fragment to be expressed. The digested vector is treatedwith calf intestinal phosphatase to prevent self-ligation. Thedephosphorylated, linear vector is fractionated on an agarose gel andpurified.

[0280] NKAF II cDNA capable of expressing active NKAF II, is isolated.The ends of the fragment are modified, if necessary, for cloning intothe vector. For instance, 5″ overhanging may be treated with DNApolymerase to create blunt ends. 3′ overhanging ends may be removedusing S1 nuclease. Linkers may be ligated to blunt ends with T4 DNAligase.

[0281] Equal quantities of the Moloney murine leukemia virus linearbackbone and the NKAF II fragment are mixed together and joined using T4DNA ligase. The ligation mixture is used to transform E. Coli and thebacteria are then plated onto agar-containing kanamycin. Kanamycinphenotype and restriction analysis confirm that the vector has theproperly inserted gene.

[0282] Packaging cells are grown in tissue culture to confluent densityin Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS),penicillin and streptomycin. The vector containing the NKAF II gene isintroduced into the packaging cells by standard techniques.

[0283] Infectious viral particles containing the NKAF II gene arecollected from the packaging cells, which now are called producer cells.

[0284] Fresh media is added to the producer cells, and after anappropriate incubation period media is harvested from the plates ofconfluent producer cells. The media, containing the infectious viralparticles, is filtered through a Millipore filter to remove detachedproducer cells. The filtered media then is used to infect fibroblastcells. Media is removed from a sub-confluent plate of fibroblasts andquickly replaced with the filtered media. Polybrene (Aldrich) may beincluded in the media to facilitate transduction. After appropriateincubation, the media is removed and replaced with fresh media. If thetiter of virus is high, then virtually all fibroblasts will be infectedand no selection is required. If the titer is low, then it is necessaryto use a retroviral vector that has a selectable marker, such as neo orhis, to select out transduced cells for expansion.

[0285] Engineered fibroblasts then may be injected into rats, eitheralone or after having been grown to confluence on microcarrier beads,such as cytodex 3 beads. The injected fibroblasts produce NKAF IIproduct, and the biological actions of the protein are conveyed to thehost.

Example 6

[0286] Expression of Recombinant NKAF II in CHO Cells

[0287] The vector pC1 is used for the expression of NKAF II protein.Plasmid pC1 is a derivative of the plasmid pSV2-dhfr [ATCC Accession No.37146]. Both plasmids contain the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Lift Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. J. and Sydenham, M.A. 1991, Biotechnology Vol.9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene it is usually co-amplified and over-expressed. It is stateof the art to develop cell lines carrying more than 1,000 copies of thegenes. Subsequently, when the methotrexate is withdrawn, cell linescontain the amplified gene integrated into the chromosome(s).

[0288] Plasmid pC1 contains for the expression of the gene of interest astrong promoter of the long terminal repeat (LTR) of the Rouse SarcomaVirus (Cullen, et al., Molecular and Cellular Biology, March 1985,438-4470) plus a fragment isolated from the enhancer of the immediateearly gene of human cytomegalovirus (CMV) (Boshart et al., Cell41:521-530, 1985). Downstream of the promoter are the following singlerestriction enzyme cleavage sites that allow the integration of thegenes: BamHI, Pvull, and Nrul. Behind these cloning sites the plasmidcontains translational stop codons in all three reading frames followedby the 3′ intron and the polyadenylation site of the rat preproinsulingene. Other high efficient promoters can also be used for theexpression, e.g., the human β-actin promoter, the SV40 early or latepromoters or the long terminal repeats from other retroviruses, e.g.,HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g.,from the human growth hormone or globin genes can be used as well.

[0289] Stable cell lines carrying a gene of interest integrated into thechromosome can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g. G418 plusmethotrexate.

[0290] The plasmid pC1 is digested with the restriction enzyme BamHI andthen dephosphorylatedusing calf intestinal phosphatase by proceduresknown in the art. The vector is then isolated from a 1% agarose gel.

[0291] The DNA sequence encoding NKAF II, 97465 is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene:

[0292] The 5′ primer has the sequence 5′ CGC +E,uns GGATCC GCC ATC ATGCAA CGC CTC TTG 3′ (SEQ ID NO:9) containing the underlined Bam HIrestriction enzyme site followed by 15 bases of the sequence of NKAF IIof FIG. 1 (SEQ ID NO:1). The 3′ primer has the sequence 5′ CGC +E,unsGGT ACC CTC CGT GCC GCT GGC TTA 3′ (SEQ ID NO:10) containing theunderlined Asp718 restriction followed by nucleotides complementary to18 nucleotides of NKAF II non-coding sequence next to stop codon.

[0293] The amplified fragments are isolated from a 1% agarose gel asdescribed above and then digested with the endonuclease BamHI and thenpurified again on a 1% agarose gel.

[0294] The isolated fragment and the dephosphorylated vector are thenligated with T4 DNA ligase. E.coli HB101 cells are then transformed andbacteria identified that contained the plasmid pC1 inserted in thecorrect orientation using the restriction enzyme BamHI. The sequence ofthe inserted gene is confirmed by DNA sequencing.

[0295] Transfection of CHO-DHFR-cells

[0296] Chinese hamster ovary cells lacking an active DHFR enzyme areused for transfection. 5 mg of the expression plasmid Cl arecotransfected with 0.5 mg of the plasmid pSVneo using the lipofectinmethod (Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the gene neo from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) and cultivated from 10-14 days. After this period,single clones are trypsinized and then seeded in 6-well petri dishesusing different concentrations of methotrexate (25, 50 nm, 100 nm, 200nm, 400 nm). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (500 nM, 1 mM, 2 mM, 5 mM). Thesame procedure is repeated until clones grow at a concentration of 100mM.

[0297] The expression of the desired gene product is analyzed by Westernblot analysis and SDS-PAGE.

[0298] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

1 11 857 base pairs nucleic acid single linear DNA (genomic) notprovided CDS 82..756 sig_peptide 82..130 mat_peptide 133..756 1CTTGGAAGGG GCTATACTAG ACACACAAAG ACAGCCCCAA GAAGGACGGT GGAGTAGTGT 60CCTCGCTAAA AGACAGTAGA T ATG CAA CGC CTC TTG CTC CTG CCC TTT CTC 111 MetGln Arg Leu Leu Leu Leu Pro Phe Leu -17 -15 -10 CTG CTG GGA ACA GTT TCTGCT CTT CAT CTG GAG AAT GAT GCC CCC CAT 159 Leu Leu Gly Thr Val Ser AlaLeu His Leu Glu Asn Asp Ala Pro His -5 1 5 CTG GAG AGC CTA GAG ACA CAGGCA GAC CTA GGC CAG GAT CTG GAT AGT 207 Leu Glu Ser Leu Glu Thr Gln AlaAsp Leu Gly Gln Asp Leu Asp Ser 10 15 20 25 TCA AAG GAG CAG GAG AGA GACTTG GCT CTG ACG GAG GAG GTG ATT CAG 255 Ser Lys Glu Gln Glu Arg Asp LeuAla Leu Thr Glu Glu Val Ile Gln 30 35 40 GCA GAG GGA GAG GAG GTC AAG GCTTCT GCC TGT CAA GAC AAC TTT GAG 303 Ala Glu Gly Glu Glu Val Lys Ala SerAla Cys Gln Asp Asn Phe Glu 45 50 55 GAT GAG GAA GCC ATG GAG TCG GAC CCAGCT GCC TTA GAC AAG GAC TTC 351 Asp Glu Glu Ala Met Glu Ser Asp Pro AlaAla Leu Asp Lys Asp Phe 60 65 70 CAG TGC CCC AGG GAA GAA GAC ATT GTT GAAGTG CAG GGA AGT CCA AGG 399 Gln Cys Pro Arg Glu Glu Asp Ile Val Glu ValGln Gly Ser Pro Arg 75 80 85 TGC AAG ACC TGC CGC TAC TAT TTG GTG CGG ACTCCT AAA ACT TTT GCA 447 Cys Lys Thr Cys Arg Tyr Tyr Leu Val Arg Thr ProLys Thr Phe Ala 90 95 100 105 GAA GCT CAG ATT GTC TGC AGC AGA TGC TACGGA GGC AAC CTT GTC TCT 495 Glu Ala Gln Ile Val Cys Ser Arg Cys Tyr GlyGly Asn Leu Val Ser 110 115 120 ATC CAT GAC TTC AAC TTC AAC TAT CGC ATCATG TGC ATC AGT AAC ACA 543 Ile His Asp Phe Asn Phe Asn Tyr Arg Ile MetCys Ile Ser Asn Thr 125 130 135 GTC AAC CAA GCC CAG GTC TGG ATT GGA GGCAAC CTC AGG GGC TGG TTC 591 Val Asn Gln Ala Gln Val Trp Ile Gly Gly AsnLeu Arg Gly Trp Phe 140 145 150 CTG TGG AAG CGG TTT TGC TGG ACT GAT GGGAGC CAC TGG AAT TTT GCT 639 Leu Trp Lys Arg Phe Cys Trp Thr Asp Gly SerHis Trp Asn Phe Ala 155 160 165 TAC TGG TCC CCA GGG CAA CCT GGG AAT GGGCAA GGC TCC TGT GTG GCC 687 Tyr Trp Ser Pro Gly Gln Pro Gly Asn Gly GlnGly Ser Cys Val Ala 170 175 180 185 CTA TGC ACC AAA GGA GGT TAT TGG CGACGA ACT CAA TGC GAC AAG CAA 735 Leu Cys Thr Lys Gly Gly Tyr Trp Arg ArgThr Gln Cys Asp Lys Gln 190 195 200 CTG CCC TTC GTC TGC TCC TTCTAAGCCAGCG GCACGGAGAC CCTGCCAGCA 786 Leu Pro Phe Val Cys Ser Phe 205GCTCCCTCCC GTCCCCCAAC CTCTCCTGCT CATAAATCCA GACTTCCCAC AGCAAAAAAA 846AAAAAAAAAA A 857 225 amino acids amino acid linear protein not provided2 Met Gln Arg Leu Leu Leu Leu Pro Phe Leu Leu Leu Gly Thr Val Ser -17-15 -10 -5 Ala Leu His Leu Glu Asn Asp Ala Pro His Leu Glu Ser Leu GluThr 1 5 10 15 Gln Ala Asp Leu Gly Gln Asp Leu Asp Ser Ser Lys Glu GlnGlu Arg 20 25 30 Asp Leu Ala Leu Thr Glu Glu Val Ile Gln Ala Glu Gly GluGlu Val 35 40 45 Lys Ala Ser Ala Cys Gln Asp Asn Phe Glu Asp Glu Glu AlaMet Glu 50 55 60 Ser Asp Pro Ala Ala Leu Asp Lys Asp Phe Gln Cys Pro ArgGlu Glu 65 70 75 Asp Ile Val Glu Val Gln Gly Ser Pro Arg Cys Lys Thr CysArg Tyr 80 85 90 95 Tyr Leu Val Arg Thr Pro Lys Thr Phe Ala Glu Ala GlnIle Val Cys 100 105 110 Ser Arg Cys Tyr Gly Gly Asn Leu Val Ser Ile HisAsp Phe Asn Phe 115 120 125 Asn Tyr Arg Ile Met Cys Ile Ser Asn Thr ValAsn Gln Ala Gln Val 130 135 140 Trp Ile Gly Gly Asn Leu Arg Gly Trp PheLeu Trp Lys Arg Phe Cys 145 150 155 Trp Thr Asp Gly Ser His Trp Asn PheAla Tyr Trp Ser Pro Gly Gln 160 165 170 175 Pro Gly Asn Gly Gln Gly SerCys Val Ala Leu Cys Thr Lys Gly Gly 180 185 190 Tyr Trp Arg Arg Thr GlnCys Asp Lys Gln Leu Pro Phe Val Cys Ser 195 200 205 Phe 26 base pairsnucleic acid single linear DNA (genomic) not provided 3 CGCCCATGGAGAATGATGCC CCCCAT 26 27 base pairs nucleic acid single linear DNA(genomic) not provided 4 CGCAAGCTTC TCCGTGCCGC TGGCTTA 27 30 base pairsnucleic acid single linear DNA (genomic) not provided 5 CGCGGATCCGCCATCATGCA ACGCCTCTTG 30 27 base pairs nucleic acid single linear DNA(genomic) not provided 6 CGCGGTACCC TCCGTGCCGC TGGCTTA 27 30 base pairsnucleic acid single linear DNA (genomic) not provided 7 CGCGGATCCGCCATCATGCA ACGCCTCTTG 30 54 base pairs nucleic acid single linear DNA(genomic) not provided 8 CGCTCTAGAT CAAGCGTAGT CTGGGACGTC GTATGGGTAGAAGGAGCAGA CGAA 54 30 base pairs nucleic acid single linear DNA(genomic) not provided 9 CGCGGATCCG CCATCATGCA ACGCCTCTTG 30 27 basepairs nucleic acid single linear DNA (genomic) not provided 10CGCGGTACCC TCCGTGCCGC TGGCTTA 27 222 amino acids amino acid singlelinear protein not provided 11 Met Lys Leu Pro Leu Leu Leu Ala Leu LeuPhe Gly Ala Val Ser Ala 1 5 10 15 Leu His Leu Arg Ser Glu Thr Ser ThrPhe Glu Thr Pro Leu Gly Ala 20 25 30 Lys Thr Leu Pro Glu Asp Glu Glu ThrPro Glu Gln Glu Met Glu Glu 35 40 45 Thr Pro Cys Arg Glu Leu Glu Glu GluGlu Glu Trp Gly Ser Gly Ser 50 55 60 Glu Asp Ala Ser Lys Lys Asp Gly AlaVal Glu Ser Ile Ser Val Pro 65 70 75 80 Asp Met Val Asp Lys Asn Leu ThrCys Pro Glu Glu Glu Asp Thr Val 85 90 95 Lys Val Val Gly Ile Pro Gly CysGln Thr Cys Arg Tyr Leu Leu Val 100 105 110 Arg Ser Leu Gln Thr Phe SerGln Ala Trp Phe Thr Cys Arg Arg Cys 115 120 125 Tyr Arg Gly Asn Leu ValSer Ile His Asn Phe Asn Ile Asn Tyr Arg 130 135 140 Ile Gln Cys Ser ValSer Ala Leu Asn Gln Gly Gln Val Trp Ile Gly 145 150 155 160 Gly Arg IleThr Gly Ser Gly Arg Cys Arg Arg Phe Gln Trp Val Asp 165 170 175 Gly SerArg Trp Asn Phe Ala Tyr Trp Ala Ala His Gln Pro Trp Ser 180 185 190 ArgGly Gly His Cys Val Ala Leu Cys Thr Arg Gly Gly Tyr Trp Arg 195 200 205Arg Ala His Cys Leu Arg Arg Leu Pro Phe Ile Cys Ser Tyr 210 215 220

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide having at least 95% identity to a member selected fromthe group consisting of: (a) a polynucleotide encoding an NKAF IIpolypeptide comprising an amino acid sequence as set forth in SEQ IDNO:2; (b) a polynucleotide encoding an NKAF II polypeptide comprisingamino acid 1 to amino acid 208 as set forth in SEQ ID NO:2; (c) apolynucleotide encoding an NKAF II polypeptide comprising amino acid 4to amino acid 208 as set forth in SEQ ID NO:2; and (d) a polynucleotidewhich is complementary to the polynucleotide of(a), (b) or (c).
 2. Thepolynucleotide of claim 1 wherein the polynucleotide is DNA.
 3. Thepolynucleotide of claim 1 wherein the polynucleotide is RNA.
 4. Thepolynucleotide of claim 1 wherein the polynucleotide is genomic DNA. 5.The polynucleotide of claim 2 which encodes the NKAF11polypeptidecomprising amino acid residues −17 to 208 of SEQ ID NO:2.
 6. Thepolynucleotide of claim 2 which encodes the mature NKAF II polypeptidecomprising amino acid residues 1 to 208 of SEQ ID NO:2.
 7. An isolatedpolynucleotide comprising a polynucleotide having at least 95% identityto a member selected from the group consisting of. (a) a polynucleotidewhich encodes the full-length NKAF II polypeptide having the amino acidsequence encoded by the human cDNA contained in ATCC Deposit No. 97465;(b) a polynucleotide which encodes the mature polypeptide having theamino acid sequence encoded by the human cDNA contained in ATCC DepositNo. 97465; (c) a polynucleotide which is complementary to thepolynucleotide of (a) or (b).
 8. The polynucleotide of claim 1comprising the sequence as set forth in SEQ ID NO:1 from nucleotide 133to nucleotide
 756. 9. The polynucleotide of claim 1 comprising thesequence as set forth in SEQ ID NO:1 from nucleotide 142 to nucleotide756.
 10. A vector comprising the DNA of claim 2 .
 11. A host cellcomprising the vector of claim 10 .
 12. A process for producing apolypeptide comprising: expressing from the host cell of claim 11 thepolypeptide encoded by said DNA.
 13. A process for producing a cellwhich expresses a polypeptide comprising genetically engineering thecell with the vector of claim 10 .
 14. A polypeptide comprising a memberselected from the group consisting of: (a) an NKAF II polypeptide havingan amino acid sequence set forth in SEQ ID NO:2; (b) an NKAF IIpolypeptide comprising amino acid 1 to amino acid 208 of SEQ ID NO:2;(c) an NKAF II polypeptide comprising amino acid 4 to amino acid 208 ofSEQ ID NO:2; and (d) an NKAF II polypeptide which is at least 95%identical to the polypeptide of (a), (b) or (c).
 15. The NKAF IIpolypeptide of claim 14 wherein the polypeptide comprises amino acid -17to amino acid 208 of SEQ ID NO:2.
 16. The polypeptide of claim 14wherein the polypeptide comprises amino acid 1 to amino acid 208 of SEQID NO:2.
 17. A compound which inhibits activation of the polypeptide ofclaim 14 .
 18. A method for the treatment of a patient having need ofNKAF II comprising: administering to the patient a therapeuticallyeffective amount of the polypeptide of claim 14 .
 19. The method ofclaim 18 wherein said therapeutically effective amount of thepolypeptide is administered by providing to the patient DNA encodingsaid polypeptide and expressing said polypeptide in vivo.
 20. A methodfor the treatment of a patient having need to inhibit a NKAF IIpolypeptide comprising: administering to the patient a therapeuticallyeffective amount of the compound of claim 17 .
 21. A process fordiagnosing a disease or a susceptibility to a disease related to anunder-expression of the NKAF II polypeptide of claim 14 comprising:determining a mutation in a nucleic acid sequence encoding saidpolypeptide.
 22. A diagnostic process comprising: analyzing for thepresence of the NKAF II polypeptide of claim 14 in a sample derived froma host.
 23. A method for identifying compounds which bind to and inhibitactivation of the NKAF II polypeptide of claim 14 comprising: contactinga cell expressing on the surface thereof a receptor for the polypeptide,said receptor being associated with a second component capable ofproviding a detectable signal in response to the binding of a compoundto said receptor, with an analytically detectable NKAF II polypeptideand a compound under conditions to permit binding to the receptor; anddetermining whether the compound binds to and inhibits the receptor bydetecting the absence of a signal generated from the interaction of theNKAF II with the receptor.